Cooperative inhibition of T-cell antigen receptor signaling by a complex between a kinase and a phosphatase.

Cloutier JF, Veillette A - J. Exp. Med. (1999)

Bottom Line:
This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains.The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling.Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association.

ABSTRACTAntigen receptor-triggered T-cell activation is mediated by the sequential action of the Src and Syk/Zap-70 families of protein tyrosine kinases (PTKs). Previously, we reported that another PTK termed p50(csk) was a potent negative regulator of T-cell receptor (TCR) signaling because of its ability to inactivate Src-related kinases. This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains. Subsequent studies uncovered that, via its SH3 domain, p50(csk) was associated with PEP, a proline-enriched protein tyrosine phosphatase (PTP) of unknown function expressed in hemopoietic cells. Herein, we have attempted to identify the role of the Csk-PEP complex in T lymphocytes. The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling. This property was dependent on the phosphatase activity of PEP, as well as on the sequence mediating its binding to p50(csk). Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association. Finally, experiments with a substrate-trapping mutant of PEP suggested that PEP functions by dephosphorylating and inactivating the PTKs responsible for T-cell activation. In addition to giving novel insights into the mechanisms involved in the negative regulation of T-cell activation, these findings indicate that the association of an inhibitory PTK with a PTP constitutes a more efficient means of inhibiting signal transduction by Src family kinases in vivo.

Mentions:
Then we examined the importance of the association of p50csk with PEP by testing the function in BI-141 cells of a PEP mutant carrying a deletion in the proline-rich region mediating binding to Csk (ΔP1 PEP; Fig. 3 A) (9, 26). While this mutation did not affect the ability of PEP to dephosphorylate the exogenous substrate myelin basic protein in vitro (Fig. 2 B), it significantly reduced (approximately fivefold) its capacity to be coimmunoprecipitated with Csk (Fig. 2 C; lanes 13–16). By opposition to our earlier finding in Cos-1 cells (9, 26), there appeared to be a partial association of ΔP1 PEP with Csk in BI-141 cells. Although the exact basis for this difference is undetermined, it is possible that other structural determinants in PEP (such as the proline-rich domain P2) mediated binding to Csk in the T-cell line. In support of this notion, we reported elsewhere that PEP P2 could also associate with the Csk SH3 domain in vitro, albeit with a greatly reduced apparent affinity in comparison with P1 (26). Alternatively, it is conceivable that the presence of ΔP1 PEP in BI-141 cells augmented by an unknown mechanism the ability of endogenous PEP molecules to associate with Csk. In any case, we observed that ΔP1 PEP failed to inhibit antigen receptor–mediated lymphokine production in BI-141 cells (Fig. 3 B), indicating that the small (approximately twofold) increase in the abundance of Csk-associated PEP in these cells was insufficient to inhibit TCR signaling. The defective function of ΔP1 PEP was reminiscent of the inability of an SH3 domain–deleted version of Csk (ΔSH3 Csk) to suppress TCR-mediated lymphokine secretion in BI-141 cells (8; Fig. 3 B). From these data, we deduced that the inhibitory effect of PEP on TCR-mediated lymphokine secretion not only requires its phosphatase activity, but also is correlated with the capacity to associate with Csk.

Mentions:
Then we examined the importance of the association of p50csk with PEP by testing the function in BI-141 cells of a PEP mutant carrying a deletion in the proline-rich region mediating binding to Csk (ΔP1 PEP; Fig. 3 A) (9, 26). While this mutation did not affect the ability of PEP to dephosphorylate the exogenous substrate myelin basic protein in vitro (Fig. 2 B), it significantly reduced (approximately fivefold) its capacity to be coimmunoprecipitated with Csk (Fig. 2 C; lanes 13–16). By opposition to our earlier finding in Cos-1 cells (9, 26), there appeared to be a partial association of ΔP1 PEP with Csk in BI-141 cells. Although the exact basis for this difference is undetermined, it is possible that other structural determinants in PEP (such as the proline-rich domain P2) mediated binding to Csk in the T-cell line. In support of this notion, we reported elsewhere that PEP P2 could also associate with the Csk SH3 domain in vitro, albeit with a greatly reduced apparent affinity in comparison with P1 (26). Alternatively, it is conceivable that the presence of ΔP1 PEP in BI-141 cells augmented by an unknown mechanism the ability of endogenous PEP molecules to associate with Csk. In any case, we observed that ΔP1 PEP failed to inhibit antigen receptor–mediated lymphokine production in BI-141 cells (Fig. 3 B), indicating that the small (approximately twofold) increase in the abundance of Csk-associated PEP in these cells was insufficient to inhibit TCR signaling. The defective function of ΔP1 PEP was reminiscent of the inability of an SH3 domain–deleted version of Csk (ΔSH3 Csk) to suppress TCR-mediated lymphokine secretion in BI-141 cells (8; Fig. 3 B). From these data, we deduced that the inhibitory effect of PEP on TCR-mediated lymphokine secretion not only requires its phosphatase activity, but also is correlated with the capacity to associate with Csk.

Bottom Line:
This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains.The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling.Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association.

ABSTRACTAntigen receptor-triggered T-cell activation is mediated by the sequential action of the Src and Syk/Zap-70 families of protein tyrosine kinases (PTKs). Previously, we reported that another PTK termed p50(csk) was a potent negative regulator of T-cell receptor (TCR) signaling because of its ability to inactivate Src-related kinases. This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains. Subsequent studies uncovered that, via its SH3 domain, p50(csk) was associated with PEP, a proline-enriched protein tyrosine phosphatase (PTP) of unknown function expressed in hemopoietic cells. Herein, we have attempted to identify the role of the Csk-PEP complex in T lymphocytes. The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling. This property was dependent on the phosphatase activity of PEP, as well as on the sequence mediating its binding to p50(csk). Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association. Finally, experiments with a substrate-trapping mutant of PEP suggested that PEP functions by dephosphorylating and inactivating the PTKs responsible for T-cell activation. In addition to giving novel insights into the mechanisms involved in the negative regulation of T-cell activation, these findings indicate that the association of an inhibitory PTK with a PTP constitutes a more efficient means of inhibiting signal transduction by Src family kinases in vivo.